Varicella-zoster virus (referred to as “varicella virus” or “VZV”) is a strictly species-specific virus, which is a pathogenicity factor that causes varicella or herpes zoster in human. The P-Oka strain of varicella virus was first isolated by Takahashi in 1971 from vesicles fluids of a 3 year-old Japanese boy of the surname Oka who has typical varicella and was obtained through passage of the virus in human embryonic lung cells. It is considered that the P-Oka has the capability of causing varicella and herpes zoster (Takahashi M, Otsuka T, Okuno Y, et al., Live vaccine used to prevent the spread of varicella in children in hospital. Lancet 1974; 2: 1288-90.).
It was disclosed in U.S. Pat. No. 3,985,615 that attenuated P-Oka strain is obtained by serial passaging of the virus in guinea pig embryonic tissue (GPEC) and human embryonic lung cells, and V-Oka live vaccine is prepared for preventing varicella. It was described in U.S. Pat. No. 4,000,256 that VZV was passaged 10-80 times in human embryonic fibroblasts WI-38 strain to produce live attenuated VZV vaccine. So far, only the “live attenuated varicella virus Oka strain” obtained by passage of the P-Oka strain in cells (special bulletin, No. 53-41202 Kimiaki), Lancet 2: 1288-1290, 1974) was approved by WHO for preparation vaccine for the prevention of varicella or herpes zoster, and the vaccine prepared from this strain has been widely used around the world [Requirement for Varicella Vaccine (Live) Adopted 1984: WHO Technical Report Series, No. 725, pp. 102-104, 1985]. Although population vaccinated with varicella vaccine have been reported to have a lower incidence of herpes zoster than populations of nature infection and most children are immonocompromised, the fact is that herpes zoster still appeared in vaccinated populations, and the V-Oka strain was also isolated from vesicles fluids of herpes zoster in vaccinated populations (Schmid D. S, et al. Impact of varicella vaccine on varicella-zoster virus dynamics, clinical microbiology reviews, January 2010, p 202-217).
The patent CN1163604C relates to the identification of the attenuated varicella vaccine Oka strain, which revealed at molecular level that the V-Oka strain is a mixed strains, which were composed of a number of different sequences. Wild strain may exists in the attenuated varicella virus strains, since it has been reported that patients inoculated with the V-Oka vaccine have developed herpes zoster containing the V-Oka strain. This means that there are still some potentially harmless in attenuated virus strains. However, so far there is no other vaccine against VZV on the market. This virus has strict species specificity, and human is the only natural host. And no animal models are available to be used in the research of the gene function of VZV. Furthermore, the virus has strong binding affinity with host cells. Free virus is easily to be inactivated. Therefore, it is very difficult to obtain VZV virus DNA that can be used for transformation. As a result, the research process of the gene function of this virus advances very slowly over a long time period.
With the application of a bacterial artificial chromosome (BAC) technology in the research, the advances of tissue culture technology and the breakthrough of barrier of organ transplant between heterogeneous organisms (the development of the combined immunodeficient mouse model), the study space of the gene function of species-specific VZV have been broaden. The study of gene functions of VZV can be carried out in vitro or it may be studied after transplanting into animals. In 2004, Kazuhiro N. et al. revealed in “Cloning of the varicella-zoster virus genome as an infectious bacterial artificial chromosome in Escherichia coli” that the whole genome of VZV Oka strain cloned into a BAC vector may proliferate in Escherichia coli and human embryonic lung cell. The BAC may be accurately excised by the enzyme Cre that is co-transformed into human embryonic lung cells. The resulted recombinant VZV virus has the same structure and characteristics as the parental virus P-Oka strain. The application of BAC has greatly facilitated the study of the gene function of VZV. It is possible to study each functional gene and thereby accelerate the research process of gene function of VZV.
The ORF7 of VZV, located at positions 8607-9386 of the viral genome, encodes a 29 kDa protein, which might locates within tegument. However, its function remains unknown.
The inventor, after extensive research, surprisingly found that ORF7 determines the function of the skin and sensory ganglion in patients infected with VZV. The ORF7 loses function when the any one of the followings occurs in ORF7: the whole or partial deletion, stop condon insertion, substituted by one or two bases, especially ORF7 deletion or flip reverse frameshift mutation. That is, VZV no longer infects human skin and sensory ganglion. So there is no potential risk of developing varicella or the risk of reactivation that causes herpes zoster (We define it here as ORF7 non-function caused by ORF7 deficiency, which function means contacting human skin and sensory ganglion). The ORF7-deletion VZV stain (VZV-7D BAC or VZV-7DRM BAC) may be further screened in E. Coli by antibiotics or galactose, in order to obtain the single clone strain. So there is no potential hazard of mixed V-Oka strains. It opens a door for developing a safer and more efficacious vaccine with ORF7 deleted or mutant virus.